The objective of this research program is to understand the interplay between the sequence and stability of proteins and their unfolding, disassembly, and degradation by cellular enzymes. These molecular processes are critical for normal cell and biological function, frequently go awry in disease, and provide potential targets for novel therapeutic strategies. Many natural proteins and assemblies are too stable for spontaneous unfolding/dissociation to be a biologically relevant reaction Energy-dependent molecular machines of the AAA+ family play critical intracellularroles in dismantling such complexes, in solubilizing aggregates, and in mediating protein degradation. To understandthese processes, we are studying the biochemical, enzymatic, and structural properties of ClpX, a hexameric AAA+ ATPase, which carries out mechanical protein denaturation and then translocates unfolded polypeptides into the ClpP protease for degradation. We have developed methods to link ClpX subunits together, allowing construction of active variants in which only a subset of the subunits of the hexamer are wild type. We will use such variants to determine how movements of loops in the central pore of ClpX mediate translocation and to determine the molecular determinants of substrates required for efficient translocation. These studies will be aided by our ability to study substrates in both native and denatured states. We will also determine structures of ClpX and HslU, a related enzyme, bound to peptide/pfotein substrates during different portions of the ATPase cycle. Knowledge of these structures should allow the substrate specificity of these molecular machines to be redesigned. ClpP has recently been shown to be the target of a new class of antibiotics in Gram-positive bacteria. Enzymes related to ClpX are virulence determinantsin some pathogenic bacteria, play roles in regulation of bio-film formation, and function in key processes in mammaliancells, including proteasome-mediated protein degradation and vesicle fusion. Hence, these basic studies have many potential health-related applications.